1. Technical Field
The present invention relates to a surface plasmon resonance sensor and a sensor chip. In particular, the present invention relates to a surface plasmon resonance sensor that uses a local surface plasmon resonance, and a surface plasmon resonance sensor chip.
2. Related Art
Human body consists of 60% water, and 40% of the remaining half protein, where the majority of the tissues, muscles, and skin of the human body consist of protein. Thus, a correlation is often recognized between the disease and the variation of the protein, where a specific protein increases in the body (e.g., in the blood) with progression of the disease in cancer, influenza, and other diseases.
Therefore, the affected site and the progressing state of the disease can be recognized by monitoring the state of a specific protein (presence, amount, etc. of a specific protein), and currently, a correlation with the disease is recognized for a few tens of types of proteins. For instance, a biological molecule that increases with progression of a tumor (cancer) is referred to as tumor marker, and different tumor markers are specified according to the site of origin of the tumor.
Biological molecules such as protein, DNA, and sugar chain in the living body are often related directly to the occurrence of disorder, and thus the mechanism of the disease can be understood and development of an effective drug can be carried out by analyzing the interaction between the biological molecules.
A tool for easily and conveniently measuring the presence and the quantity of a specific protein including the tumor marker at high accuracy includes a biosensor, and application to false diagnosis prevention, early diagnosis, preventive medical care, and the like is expected in the future.
A method of detecting the interaction of the biological molecules such as protein uses surface plasmon resonance. The surface plasmon resonance (SPR) is a resonance phenomenon that occurs by interaction between free electrons of the metal surface and electromagnetic wave (light), and is given attention as an easy and convenient method since the sample does not need to be labeled with fluorescent substance compared to the fluorescent detecting method. A sensor using surface plasmon resonance includes a propagation surface plasmon resonance sensor and a local surface plasmon resonance sensor.
The principle of the propagation surface plasmon resonance sensor will be briefly described with FIGS. 1A to 1D. As shown in FIGS. 1A and 1C, the propagation surface plasmon resonance sensor 11 has a metal film 13 made of Au, Ag, and the like having a thickness of about 50 nm formed on the surface of a glass substrate 12. The propagation surface plasmon resonance sensor 11 irradiates light from the glass substrate 12 side, and totally reflects the light at an interface of the glass substrate 12 and the metal film 13. The biological molecule or the like is sensed by receiving the totally reflected light, and measuring the reflectance of the light.
In other words, when carrying out reflectance measurement by changing the incident angle θ of the light, the reflecting angle greatly attenuates at a certain incident angle (resonance incident angle) θ1, as shown in FIG. 1B. When the light entering the interface of the glass substrate 12 and the metal film 13 is totally reflected at the relevant interface, evanescent light (near-field light) generated at the relevant interface and the surface plasmon wave of the metal interact. At a specific wavelength or a specific incident angle, the energy of the light is absorbed into the metal film 13 and changed to vibration energy of the free electrons in the metal film 13, whereby the reflectance of the light significantly lowers.
The resonance condition depends on the dielectric constant (index of refraction) of the peripheral substances of the metal film 13, and thus is used for a method of detecting change in characteristics of the peripheral substances at high sensitivity. In particular, when used as a biosensor, an antibody 14 (probe) that uniquely bonds with a specific protein (antigen) is immobilized on the surface of the metal film 13 in advance, as shown in FIG. 1A. If an antigen 16 that acts as a target is present in an introduced test sample 15, the antigen 16 and the antibody 14 uniquely bond, as shown in FIG. 1C. The index of refraction around the metal film 13 changes when the antigen 16 is bonded, and the resonance wavelength and the resonance incident angle change. Therefore, whether or not the antigen 16 is contained in the test sample 15 can be examined by measuring change in the resonance wavelength, change in the resonance incident angle, or temporal change in the resonance wavelength and the resonance incident angle at before and after introducing the test sample 15. At what extent of the concentration the antigen 16 is contained can also be examined.
FIG. 1D shows one example of a result of measuring the dependency of reflectance with respect to the incident angle θ. In FIG. 1D, a broken line shows a reflectance spectrum 17a before the test sample 15 is introduced, and a solid line shows a reflectance spectrum 17b after the test sample 15 is introduced and the antigen 16 is bonded to the antibody 14. Whether or not the test sample 15 contains the antigen 16 can be examined by measuring the change Δθ in resonance incident angle at before and after the test sample 15 is introduced. Furthermore, the concentration of the antigen 16 can also be examined, and the presence of a specific pathogenic agent, presence of disorder, and the like can be examined.
A prism is used to introduce light to the glass substrate in a general propagation surface plasmon resonance sensor. Thus, the optical system of the sensor becomes more complicating and larger, and furthermore, the sensor chip (glass substrate) and the prism need to be closely attached with matching oil. In order to resolve the difficulty in handling, Japanese Laid-Open Patent Publication No. 2002-357543 proposes a propagation surface plasmon resonance sensor that uses diffraction grating.
The propagation surface plasmon resonance sensor disclosed in Japanese Laid-Open Patent Publication No. 2002-357543 has a metal film 19 formed on a substrate 18, and a thin film discretely stacked on the metal film 19 to form a diffraction grating 20 (grating), as shown in FIG. 2.
In such propagation surface plasmon resonance sensor, when light is irradiated onto the diffraction grating 20, the irradiated light is reflected by the diffraction grating 20, and the evanescent light is generated by the diffraction phenomenon. The surface plasmon wave generates at the surface of the diffraction grating 20 when light is irradiated onto the diffraction grating 20. The evanescent light and the surface plasmon wave resonate thereby generating surface plasmon resonance when light of a certain wavelength is irradiated at a specific incident angle. Therefore, a specific antigen can be detected in such propagation surface plasmon resonance sensor by immobilizing the antibody on the diffraction grating 20.
The cross section of the diffraction grating type propagation SPR sensor may be a rectangular shape, but in most cases, the cross section is generally a sinusoidal wave shape. The concave-convex part in the diffraction grating type propagation SPR sensor serves as diffraction grating, and thus the duty ratio thereof is desirably 1:1.
FIG. 3 shows a diffraction grating of concave-convex shape. Assume the pitch of the diffraction grating 20 is P, that is, the width of the convex part (light reflecting surface) is P/2, the width of the concave part as P/2, the incident angle of the incident light as θ, and the exit angle of the diffracted light is φ. The optical path difference of the light reflected at adjacent convex parts is, as apparent from FIG. 3,P cos θ−P cos φTherefore, the condition the phases of the reflected lights align and strengthen each other is,cos θ−cos φ=m·λ/P where λ is the wavelength of the incident light. m=0, ±1, . . . is the diffraction order. The maximum value of the left side of the equation is 2, and thus (λ/P)<2 is necessary to cause diffraction.
In the diffraction grating type propagation SPR sensor, P>400 nm is a prerequisite as a condition for causing diffraction at the entire visible light region when using the light of visible light region (λ=400-800 nm) for a sensor.
In the diffraction grating type propagation SPR sensor, the light is generally entered in a diagonal direction.
Consequently, the diffraction grating type SPR sensor includes a wide sensing area of a few hundred nm.
The local surface plasmon resonance sensor will now be described. The local surface plasmon resonance sensor has the free electron vibration caused by the surface plasmon resonance standing in the local region of the metal nano configuration, and the sensing region thereof is known to be very small of a few dozen nm compared to the propagation surface plasmon resonance sensor.
The local surface plasmon sensor includes that disclosed in Japanese Laid-Open Patent Publication No. 2000-356587. The local surface plasmon resonance sensor 21 realizes the metal nano configuration using metal fine particles (metal nano fine particles) having an average particle diameter of a few dozen nm. In the sensor 21, a glass substrate 22 is immersed in colloidal solution of metal fine particles, and metal fine particles 23 are distributed over the surface of the glass substrate 22. As shown in FIG. 4A, the light is perpendicularly entered from the back surface side of the glass substrate 22, and the intensity of the light transmitted through the glass substrate 22 is measured. The received transmitted light is divided by a spectroscopic means to obtain absorbance at each wavelength, so that a peak of the absorbance is found at a certain wavelength (resonance wavelength), as shown in FIG. 4B.
The resonance phenomenon is such in which the free electrons in the metal fine particles 23 vibrate by alternating current electric field of the light (electromagnetic wave), the light and the free electrons vibrate at a certain vibration frequency so that the free electrons absorb the light energy, and the absorbance peaks at the resonance wavelength.
Since such phenomenon is also influenced by dielectric constant (index of refraction) of the periphery of the metal fine particles 23, the absorbance changes when some kind of dielectric substance (e.g., antigen) attaches to the metal fine particle. For instance, the absorbance characteristic 24a shown with a broken line in FIG. 4B before the dielectric substance attaches to the metal fine particle changes to the absorbance characteristic 24b shown with a solid line in FIG. 4B after attachment. Therefore, the presence of attachment and the quantity of attachment of the dielectric substance can be detected by reading change in peak value of the absorbance.
The surface plasmon resonance sensor includes propagation surface plasmon resonance sensor and local surface plasmon resonance sensor as described above. However, the sensing area is large or a few hundred nm from the surface of the glass substrate compared to the size of the protein (around ten nm) in the propagation surface plasmon resonance sensor. Thus, the sensor is likely to be influenced by temperature change of the test sample and foreign substances (e.g., protein other than the testing target) in the test sample, and is also sensitive to antigen that floats in the test sample without being bonded to the antibody in biosensors. These become the cause of noise, and thus it becomes difficult to manufacture a sensor having small S/N ratio and high sensitivity. In order to manufacture a sensor of high sensitivity, a step of removing foreign substances that become the cause of noise, and a strict temperature controlling means for maintaining the temperature of the test sample constant are required, whereby the device becomes larger and the device cost becomes expensive.
In the local surface plasmon resonance sensor, on the other hand, the near-field that generates at the surface of the metal fine particles (metal nano fine particles) becomes the sensing region, and thus a sensitivity region of a few dozen nm of lower than or equal to a diffraction limit can be achieved. As a result, the local surface plasmon resonance sensor is not sensitive to testing objects that float in the region distant from the metal fine particles, and is sensitive only to the testing object attached to a very narrow region of the surface of the metal fine particle surface, and thus a sensor of high sensitivity can be realized. According to such sensor, a strict temperature controlling means for controlling the temperature of the test sample to a constant temperature is not necessary.
The local surface plasmon resonance sensor using metal fine particles is not sensitive to testing object floating distant from the metal fine particles, and thus the noise component reduces, and high sensitivity is obtained in such regards compared to the propagation surface plasmon resonance sensor. However, in sensors using surface plasmon resonance generated at the metal fine particles such as Au and Ag, the intensity of the signal obtained from the testing object attached to the surface of the metal fine particles is small, and thus the sensitivity is still low or the sensitivity is not sufficient in such regards.
“Investigation Report on Metal Nano Particle Interaction and Biosensor” (FIG. 2) by Takayuki Okamoto, Plasmonic study group, 2002 Grants in Aid for Scientific Research (basic research C), research progress report; (http://www.plasmon.jp/index.html) is a research paper written by the inventor of Japanese Laid-Open Patent Publication No. 2000-356587. FIG. 5 shows a view cited from “Investigation Report on Metal Nano Particle Interaction and Biosensor” (FIG. 2) by Takayuki Okamoto, Plasmonic study group, 2002 Grants in Aid for Scientific Research (basic research C), research progress report; http://www.plasmon.jp/index.html (FIG. 2), showing “absorption spectrum of when the glass substrate deposited with gold nano particles having an average diameter of 20 nm is immersed in a liquid having various indexes of refraction”. The change in the sensitivity of the sensor, that is, the resonance wavelength in a case of index of refraction of a medium contacting the sensor changed by one is obtained based on FIG. 5. The sensitivity is about 100 nm/RIU, and a sufficient sensitivity cannot be obtained.
It is noted that from FIG. 5, such sensitivity value was calculated by reading the change in resonance wavelength as 30 nm when the index of refraction of the medium was changed from 1.333 (water) to 1.737 (diiodemethane).Sensitivity=30/(1.737−1.333)=74 [nm/RIU]The sensitivity of the sensor was about 100 nm/RIU approximating to be slightly larger than such value.
A method of immobilizing the metal fine particles on the glass substrate by immersing the glass substrate in a colloidal solution is adopted in the local surface plasmon resonance sensor of Japanese Laid-Open Patent Publication No. 2000-356587, where the distribution density of the metal fine particles is not constant even if manufactured with the same process and the variation in the distribution density is large. Furthermore, the metal fine particles can be relatively easily separated if the distribution density of the metal fine particles is small, as shown in FIG. 6A, but the metal fine particles condense as shown within a circle in FIG. 6B if the distribution density of the metal fine particles becomes large, and it becomes difficult to stabilize the quality. Therefore, a problem of mass production arises when commercializing, and in particular, mass productivity becomes worse when the distribution density of the metal fine particles is increased to enhance the sensitivity.
N. Felidj et al. “Controlling the optical response of regular arrays of gold particles for surface-enhanced Raman scattering”, Phys. Rev. B 65, 075419 (2002) shows a method of manufacturing a metal nano configuration in which the metal fine particles are evenly arrayed using an electron beam. This method will be described according to FIGS. 7A to 7F. First, as shown in FIG. 7A, a photoresist 26 is coated on the entire surface of a substrate 25. Then, as shown in FIG. 7B, an electron beam 27 is sequentially irradiated onto the photoresist 26 in a metal fine particle forming device on the substrate 25 to solubilize the photoresist 26 at the irradiating position of the electron beam 27. The photoresist 26 is subjected to development process, whereby, the solubilized portion of the photoresist 26 is removed, and a hole 28 is opened at the metal fine particle forming position in the photoresist 26, as shown in FIG. 7C. As shown in FIG. 7D, Cr29 is then deposited from above the photoresist 26, so that Cr29 is also deposited on the surface of the substrate 25 exposed in the hole 28. Thereafter, as shown in FIG. 7E, Au30 is deposited from above the photoresist 26, so that Au30 is also deposited on the Cr29 in the hole 28. The substrate 25 is thereafter immersed in the etchant to strip the photoresist 26, whereby a metal fine particle (Au30) is formed in a desired pattern at the surface of the substrate 25, as shown in FIG. 7F. FIG. 8 shows a view showing a pattern of the metal fine particle obtained in this manner (cited from N. Felidj et al. “Controlling the optical response of regular arrays of gold particles for surface-enhanced Raman scattering”, Phys. Rev. B 65, 075419 (2002), where the portion that appears white is the metal fine particle.
According to the method of FIG. 7, the metal fine particle can be formed with an arbitrary arrangement pattern, but it takes a few hours in the step of irradiating the electron beam since the pattern is drawn one at a time with the squeezed electron beam. Therefore, about half a day is required to manufacture one sensor chip, and the manufacturing throughput is very low. Therefore, mass production is difficult with such method, and realization of an inexpensive sensor chip is impossible.